Heterologous expression of a 2-substituted-(1-3)-B-D-glucan in Lactococcus lactis

MARÍA LAURA WERNING,; PILAR FERNÁNDEZ DE PALENCIA; MARÍA DE LOS ANGELES CORRALES; ALICIA PRIETO; PALOMA LÓPEZ

Egmond an Zee

Simposio; IX SYMPOSIUM ON LACTIC ACID BACTERIA: HEALTH, EVOLUTION AND SYSTEMS BIOLOGY; 2008

Abstract Exopolysaccharides (EPS) play an important role in the rheology and texture of dairy and oat-based foods and of these β-glucans have prebiotic properties. In a previous study the plasmidic gtf gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider was cloned and sequenced. The GTF product belongs to the COG1215 membrane-bound glycosyltransferase family and has identity (33%) only with the Tts glycosyltransferase of Streptococcus pneumoniae serotype 37. It catalyzes the biosynthesis and secretion of this organism’s capsule, which is a β-D-glucan similar to the EPS synthesized by Pediococcus. In this work, gtf was cloned under control of the PnisA promoter (plasmid pNGTF) and GTF was functionally expressed in Lactococcus lactis NZ9000. Agglutination tests using anti-serotype 37 antibodies and electron microscopy revealed that the enzyme confers to L. lactis NZ9000[pNGTF] the ability to synthesize and secrete EPS. GTF glycosyltransferase activity was detected by testing the membrane-bound protein with UDP-glucose substrate. The EPS released to the medium by NZ9000[pNGTF] was purified from the supernatant by centrifugation and repeated ethanol precipitation. The EPS was structurally characterized by monosaccharide, methylation and NMR analysis. The results revealed that the EPS synthesized by L. lactis NZ9000[pNGTF] was the same as that of P. parvulus 2.6. Expression in a defined medium, coupled with the purification method described here, allows approximately 80% recovery of pure β-D-glucan with a yield of 300 mg L-1 and apparent molecular mass of >1000 kDa, as determined by analysis in a Sepharose CL-6B column. Consequently, L. lactis NZ9000[pNGTF] is a convenient source of 2-substituted-(1 3)-β-D-glucan with potential utility as a food additive